Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 μg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.Under hypoxic conditions, the expression of  Solanaceae, including Hyoscyamus, Duboisia, Atropa, and Scopolia, produce biologically active tropane alkaloids (TAs)1. TAs are a class of alkaloids characterized by the presence of a bicyclic nitrogen bridge across a seven-carbon ring. The biosynthesis of hyoscyamine and scopolamine is initiated by decarboxylation of the nonproteinogenic amino acid, ornithine3. The conversion of scopolamine, which is the 6,7-β-epoxide of hyoscyamine, is catalyzed by hyoscyamine 6β-hydroxylase  via two consecutive steps: hydroxylation and epoxidation. In addition, it is known that catalysis of the final reaction in the epoxide formation requires an alkaloid substrate, 2-oxoglutarate, Fe2+, molecular oxygen, and ascorbate  is characterized as an oxygen carrier and transporter with extraordinarily high koff rate for oxygen release11. The oxygen-dependent VHb promoter was reported to be maximally induced under microaerobic conditions (with dissolved oxygen levels being less than 2% of the air saturation) in its native host13. The expression of VHb leads to the promotion of host growth and improvement in the production of some metabolic products16. The functional investigation of VHb suggested that it might increase the activity of terminal oxidases by increasing the local supply of oxygen and by shifting the cellular physiology to the energetically more efficient aerobic state19.Nicotiana tabacum plants expressing VHb were reported to grow faster and had a shorter lifecycle than those of the control plants. A shift in secondary metabolite synthesis from anabasine to nicotine, the synthesis of which is more O2-demanding, was also observed in the transgenic tobacco plants20. The VHb-producing cabbage seeds germinated more rapidly than those of the control plants21. Previously, we conducted similar studies employing the expression of VHb for the optimization of plant metabolism. The VHb gene was first introduced into Arabidopsis thaliana to investigate its positive effects on the metabolism in Arabidopsis. The constitutive expression of VHb improved the germination rate of transgenic seeds and affected the levels of some endogenous genes of Arabidopsis that are involved in oxygen metabolism and biosynthesis of antioxidants. Moreover, the VHb transgenic plants were more tolerant to photo-oxidative damage because of the increased production of antioxidants, such as ascorbate16. These results suggested that the VHb gene might potentially be used in molecular breeding for improving the production of TAs in H. niger hairy roots.The beneficial effects of heterologous expression of VHb in plants have been known for more than a decade. 23. Moreover, recent evidence has revealed that VHb is localized to the cytoplasm and is concentrated near the cytosolic face of the cell membrane24. Compartmentalization engineering is a promising strategy to increase the productivity of a desired secondary metabolite in plants25; for example, Kappers et al. introduced a linalool/nerolidol synthase into the mitochondria of Arabidopsis by fusing a bona fide mitochondrial targeting signal-CoxIV (cytochrome oxidase subunit IV) sequence from yeast. They reported that mitochondrial targeting of the sesquiterpene synthase resulted in the emission of two new isoprenoids from the A. thaliana plants and aided in their defense mechanism26. This prompted us to investigate the consequences of subcellular targeting of VHb on the production of TAs in the hairy roots of H. niger.The active regulation of VHb within the metabolic network in each cell is also determined by the enzymes expressed in the cell. For example, localization of VHb to different cellular compartments  may result in phenotypic differences in the hostH. niger with the aim of enhancing the production of TAs. The effects of VHb expression on the accumulation of hyoscyamine and scopolamine and on the transcription of TA biosynthesis genes were also investigated. Our results demonstrate that targeted VHb expression significantly increased the TA content in the H. niger hairy roots, and resulted in the up-regulation of key genes involved in the TA biosynthetic pathway. Overall our results prove that targeted expression of VHb is a simple and efficient approach to increase the production of TAs in the hairy roots of H. niger.In this study, we targeted the expression of VHb in the mitochondria and plastids of the hairy roots of H. niger, a 20-amino acid (AA) N-terminal mitochondrial localization signal from subunit IV of yeast CoxIV, an N-terminal extension with features characteristic of mitochondrial and chloroplast transit peptides from Arabidopsis (AtHRS1)27, and an A. thaliana gene that encodes a Mg2+- and ATP-dependent RecA homologue targeted to plastids28, were selected, respectively. To experimentally verify the subcellular localization of VHb, RecA, CoxIV, and AtHRS1, four binary plant expression fusion constructs, namely pVHb, pVHb-CoxIV, pVHb-RecA, and pVHb-AtHRS1 were generated in the pCAMBIA1301-GFP background. The pCAMBIA1301-GFP construct served as a control. Plastid signals were observed by the autofluorescence of chlorophyll. To mark the mitochondria, a translational fusion construct of the mitochondrial F1ATPase γ-subunit and the red fluorescent protein (RFP)29 was co-transformed into the protoplasts. As shown in Fig. pVHb-RecA was exclusively localized to the plastids. However, the CoxIV-VHb-GFP signal co-localized with the F1ATPase-γ-RFP signal but not obviously with the chlorophyll autofluorescence signal , 22 pVHb-AtHRS1 (A), 20 pVHb-CoxIV (C), 20 pVHb-RecA (R), 17 CK, and 15 WT hairy root lines were harvested, of which 11 V, 9 A, 10 C, 9 R, 11 CK, and 13 WT lines survived the successive subculture process. From these, every eight lines were selected for RNA extraction, metabolite analysis and qRT-PCR analysis . All the hairy root lines entered a stagnant phase after 45 days  among the WT, CK, and pVHb, 22 pVHb-AtHRS1, 20 pVHb-CoxIV, and 20 pVHb-RecA transgenic lines.The morphology and growth rate of the hairy root lines were evaluated by determining the increase in fresh weight (FW). WT, VHb-expressing, and CK lines showed a similar growth curve Fig. . In shakays Fig. . The difpCAMBIA1301-GFP transgenic lines than in WT. The accumulation of TAs was stimulated by the expression of VHb in the H. niger hairy roots and the increase in the hyoscyamine content varied from 2.5- to 3.6-fold in the four transgenic lines with respect to the content in the non-transgenic lines (53.91 μg/g DW) ; it was 1.25-fold of that in the lines in which VHb was expressed in a non-targeted manner (pVHb). The increase in the content of scopolamine varied from 2.2- to 4.7-fold in the four transgenic lines with respect to the content in the non-transgenic lines (64.38 μg/g DW) .The contents of hyoscyamine and scopolamine in the WT, CK, and transgenic hairy root lines were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The production of TAs could be detected in both the transgenic and non-transgenic hairy roots but varied in the different lines Fig. . InteresPMT and H6H genes, which encode the rate-limiting enzymes that can be genetically modified in H. niger plants. Previously, simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme PMT and the downstream enzyme H6H in transgenic H. niger hairy root cultures was reported7. We concluded that transgenic plants harboring both PMT and H6H had an increased flux through the TA biosynthetic pathway that enhanced the yield of scopolamine, which was more efficiently produced than in the plants harboring only one of these genes. Moreover, studies have shown that cytochrome P450 can catalyze a series of oxidation reactions, and the evolution of oxygen occurs mainly in the chloroplasts32. Therefore, the expression levels of the three genes in the TA biosynthetic pathway, namely PMT, H6H, and CYP80F1, were determined using qRT-PCR to study the effects of VHb on transcription. The transcription levels of PMT and H6H increased marginally in the pVHb-RecA line, whereas in the pVHb-CoxIV and pVHb-AtHRS1 lines, the expression levels were similar to those in the control lines. In comparison to the CK-transformed control lines, the transcript levels of CYP80F1 in the pVHb, pVHb-RecA, pVHb-CoxIV, and pVHb-AtHRS1 lines were coordinately enhanced . The highest CYP80F1 expression was observed in the pVHb-RecA lines, which showed a 4-fold increase compared to the expression levels in the non-transgenic lines, an observation that was consistent with the increase in the content of TAs  35S promoter. The expression of VHb in the plastids significantly enhanced the yield of TAs in comparison to their content in transgenic lines expressing this gene in a non-targeted manner and in the control lines. Our research, evaluating the expression of TA pathway genes and secondary metabolism in genetically modified lines, could lead to a more in-depth understanding of the function of heterologous VHb.In summary, heterologous VHb was expressed in the hairy roots of H. niger seeds were obtained from the Plant Garden of Shenyang Pharmaceutical University, Shenyang, China. To obtain sterilized seedlings, the H. niger seeds were surface-sterilized in 75% ethanol for 3 min, washed five times with distilled water, and then treated with sodium hypochlorite solution, containing 33% active chlorine, for 4 min. After thorough rinsing five times with sterile distilled water, the sterilized seeds were incubated between several layers of sterilized wet filter paper and placed on MS basal medium , the pH of which was adjusted to 5.8–6.0, for germination. The culture room was maintained at a temperature of 25 °C; the light intensity was 350 μmol. m−2 s−1, provided by white fluorescent tubes in 16-h photoperiods, and the relative air humidity was maintained at 30%. Sterile plants of H. niger were prepared as described above and 2-month-old seedlings were used for the Agrobacterium-mediated transformation experiments.NcoI and BglII restriction sites, were synthesized by GENWIZ Biotechnology . The addition of the remaining two localization sequences was done as follow: Arabidopsis RecA and yeast CoxIV were PCR-amplified . For the pCAMBIA1301-GFP-VHb-AtHRS1 (pVHb-AtHRS1), pCAMBIA1301-GFP-VHb-RecA (pVHb-RecA), and pCAMBIA1301-GFP-VHb-CoxIV (pVHb-CoxIV) binary expression vectors, the AtHRS1, RecA, and CoxIV fragments were subcloned into the pVHb vector, generating plasmids pVHb-AtHRS1, pVHb-RecA, and pVHb-CoxIV, respectively  and a 233-bp fragment of the AtHRS1 gene, containing the localization sequences with ely Fig. . The clopVHb, pVHb-CoxIV, pVHb-RecA, and pVHb-AtHRS1 vectors that contained the GFP tag were transformed into freshly prepared Oryza sativa protoplasts using PEG-mediated transformation; this was followed by transformation with the control mitochondrial F1ATPase-g-RFP fusion construct 29.The transfected protoplasts were incubated overnight at room temperature and transient expression was visualized using a confocal laser scanning microscope  as described by Tan46. The GFP fluorescence was visualized using an excitation wavelength of 488 nm and emission wavelengths in the range from 505 to 530 nm. The red autofluorescence of chlorophyll was captured at an emission wavelength longer than 650 nm. The captured images were processed using Photoshop software.The H. niger plants was carried out following a previously described method1, and simultaneous transformation with C58C1 strains was done to serve as the wild-type control47. The culture of H. niger hairy roots was obtained after the infection of leaf explants with C58C1 and these roots were developed at the cut edges after 1–2 week of co-cultivation. They were then excised and cultured on solid, hormone-free, half-strength MS medium supplemented with 30 g/L sucrose, which served as the carbon source42. All the culture media contained 100 mg/L hygromycin and 500 mg/L cefotaxime. The rapidly growing hygromycin-resistant lines, without bacterial contamination, were used to establish the hairy root lines. Approximately 100 mg of fresh roots were inoculated into 250 mL conical flasks containing 200 mL of liquid half-strength MS medium, cultured on an orbital shaker (110 rpm) at 25 °C in the dark, and the culture medium was refreshed every nine days. The FW of root tissues from the culture flasks was recorded individually after 9, 18, 27, 36, and 45 days of inoculation. At day 45, the harvested hairy roots (0.1 g) were used for DNA and RNA isolation; the remaining hairy roots were separated from the culture medium by filtration, washed three times with distilled water, blotted dry on paper towels to yield the FW, and then dried at 45 °C in an oven until a constant DW was obtained. The experiment was performed in triplicate, and the results presented are means ± S.E.M.The transformation of leaf explants from the pVHb, pVHb-AtHRS1, pVHb-RecA, and pVHb-CoxIV genes was examined using PCR with the primers shown in Supplementary Table VHb, AtHRS1, CoxIV, and RecA were specially designed so as to cover both the gene and the vector sequences. The selectable marker gene, hygromycin phosphotransferase (hpt), was used to confirm the empty vector transformants, whereas the Agrobacterium rolB and rolC genes were used to confirm the transformation of pRiA430.The presence of the transformed H. niger transgenic lines and control lines were mixed with 5X sample loading buffer , heated to 100 °C for 5 min, loaded onto a 10% SDS-polyacrylamide gel, and electrophoresed at 80 V for 120 min in a Bio-Rad Mini Trans-Blot cell. The total protein was subsequently transferred to PVDF membrane. The membranes were blocked using 5% non-fat dry milk in PBS-0.05% Tween-20 and incubated overnight with the primary antibody  at a 1:5000 dilution in 3% Bovine serum albumin. The blots were washed three times with TBST ) and incubated for 4 h with goat anti-mouse IgG antibody (1:5000) . The bands were developed using a chemiluminescent substrate .The fresh hairy root samples were homogenized in 500 μL of plant protein extraction reagent containing 1X protease inhibitor cocktail, according to the manufacturer’s recommendation . The whole protein extracts from the Kamada et al.48, with some modifications. The hairy roots were collected and dried at 45 °C in an oven until a constant DW was attained. They were then powdered finely and 50 mg of the powdered sample was sonicated in polypropylene test tubes with an extraction solvent consisting of methanol:ammonia  (4 mL) for 45 min; when fully oscillated, 6 mL chloroform was added and the mixture was kept at room temperature overnight. After freeze-drying, the residue was dissolved in 2 mL of methanol , and then centrifuged at 10,000 × g for 5 min. The supernatant was diluted ten times with methanol, and the extract was filtered through a 0.2-μm filter membrane before analysis.The extraction of tropane alkaloids (hyoscyamine and scopolamine) was based essentially on the method described by 2 = 0.9993) and from 1 to 3000 ng/mL (R2 = 0.9996), respectively.The contents of hyoscyamine and scopolamine were determined by LC-MS/MS. The analysis was performed on an Agilent 1200 infinity LC column coupled with an Agilent 6410 Triple Quadrupole LC/MS System . The LC operating conditions were as follows: LC column, Agilent ZORBAX SB-C18, 2.1 × 100 mm, 3.5 μm silica; mobile phase: acetonitrile/0.051% formic acid with 5 mM sodium acetate (32:68); total flow rate of mobile phase, 0.3 mL/min; run time, 1.8 min. The injection volume was 2 μL. The MS/MS was operated with an electrospray ionization source in positive ion mode. The pressure of the nebulizer gas was set at 40 psi, the source temperature was 350 °C, and the flow rate of gas was 10 L/min. The capillary voltage was 4000 V (positive mode). High-purity nitrogen gas was used as the collision cell gas. The raw chromatograph and the mass spectrogram data were processed with the Mass Hunter Workstation software . Multiple reaction monitoring mode (MRM) was used for quantification, and the selected transitions of m/z were 290.3R124.2 for hyoscyamine and 304.21R138.2 for scopolamine . The quality and concentration of RNA were examined by NanoDrop  and by visualizing the bands on ethidium bromide-stained agarose gels. The total RNA (1 μg) was reverse-transcribed to obtain cDNA using TransScript First-Strand cDNA Synthesis SuperMix Kit , according to the manufacturer’s instructions. qRT-PCR was performed following the instructions from the SYBR-Green PCR Master Mix Kit  on a Thermal Cycler Dice Real Time System TP800 . An efficiency-corrected comparative Ct method49 was applied and the relative expression of genes was calculated by normalizing the expression of the genes of interest to the abundance of the housekeeping gene . All the qRT-PCR experiments were performed in three independent replicates. All the primers used for qRT-PCR are shown in Supplementary Table The qRT-PCR analysis was performed to determine the transcript abundance of p < 0.05, to determine significant differences between the means.The experiments were performed in triplicate, and all the results are expressed as means ± SEM. All statistical analysis was performed using One-Way ANOVA., which was followed by Tukey’s pairwise comparison test, at a level of supplementary information


The bacterial isolates were identified using phenotypic and genotypic analyses. The antibiotic susceptibility profile of the isolates was determined by disk diffusion. PCR was used to determine the frequency of β-lactamase genes.Escherichia coli, Staphylococcus epidermidis and Klebsiella pneumoniae were the most frequent isolates in the peripheral blood cultures. E. coli and Stenotrophomonas maltophilia were isolated from two UCB cultures. The highest resistance among the Gram-positive strains was to cefixime, ceftriaxone, cefotaxime and clindamycin. In the Gram-negative bacteria the highest rates of resistance were to ampicillin (91.7%). The frequency of blaOXA-48 and blaCTX-M-15 genes was 25% and 50%, respectively.Among the 153 infants, 21 (13.7%) proved positive for sepsis. The high antibiotic resistance among the isolates reveals the importance of monitoring antibiotic consumption and improving control standards in the health care system, especially in neonatal wards. Streptococci (GBS) in developed countries  and Late-Onset Sepsis (LOS). EOS is usually caused by vertical transmission and appears during the first three days after childbirth, while horizontal transmission by hand/body contact or intra-venous catheters can lead to LOS within four to 28 days of birth . Despiteountries . The useblaOXA-48, have been of great interest in recent years owing to their ability to hydrolyze carbapenems and spread extensively among Enterobacteriaceae strains  and Oxacillin-hydrolyzing β-lactamase . Today,  strains . Moreove strains . Given ablaCTX-M-15 (Cefotaximase-M-15) and blaOXA-48 (Oxacillinase-48) resistant genes in Gram-negative isolates at Shahid Beheshti-hospital in Isfahan, Iran.The present study was designed to assess the frequency and origin of neonatal infection and determine the antibiotic resistance pattern with an emphasis on the frequency of From May 2017 to June 2018, UCB samples were collected from neonates at a teaching hospital of Isfahan, who met at least two of the following criteria: Premature birth (less than 37 weeks), prolonged rupture of the membranes (18 hours), prolonged delivery (more than 24 hours) and multiple vaginal examinations. Peripheral blood samples (PB) of the newborns with even one symptom of sepsis were also examined. A total of 153 peripheral blood samples and 108 UCB samples were collected from the neonates. Three different peripheral blood samples were taken from each neonate. The first sample was taken at the onset of fever, the second one was taken 60 min later and the third one 24 h later.2 for 24–48 h.At first, all the samples were cultured in BACTEC medium . They were then cultured on blood agar (5% sheep blood), eosin methylene blue (EMB) and chocolate agar. The plates were incubated at 37°C with 5% COThe Gram-positive isolates were identified using biochemical tests such as the catalase test, the coagulase test, Novobiocin susceptibility assay, arabinose fermentation, the bile esculin test and the CAMP test, while the IMVIC, urea, TSI, SIM, LIA and OF tests and oxidase fermentation were used for evaluating the Gram-negative strains . 16S rRNThe antibiotic resistance assay was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines using the agar disc diffusion method (DDM) for vancomycin (30 mg), ampicillin 10 mg), ciprofloxacin (5 mg), ceftriaxone (30 mg), meropenem (10 mg), colistin (10 mg), cefoxitin (30 mg), cefotaxime (30 mg), cefixime (5 mg), amoxy clavulanic acid (20/10 mg), gentamicin (10 mg), erythromycin (30 mg), clindamycin (2 mg) and penicillin (10 mg)  ;, and those that showed positive results in this test were used for the detection of  primers . DNA was2 test and Fisher’s exact test. P<0.05 was regarded as statistically significant.The measurements were reported as mean ± standard deviation (SD) for the quantitative variables and percentage or rate for the categorical variables. Inter-group comparisons were conducted in SPSS-22 using the χA total of 153 peripheral blood and 108 UCB samples were investigated during the 13 months of the study. The neonates’ age ranged from 1 to 28 days (mean =4.7±6.9 days). The blood culture results confirmed sepsis in 21 neonates, including nine (43%) boys and 12 (57%) girls, with a 1.3:1 girl/boy ratio. The youngest neonate was a day old and the oldest 20 days old. 2 test showed that leukopenia is significantly higher in neonates with EOS, while leukocytosis was higher among the LOS neonates (P=0.02). The χ2 test also demonstrated no relationship between the frequency distribution of gender and the prevalence of LOS or EOS (P=0.58).Klebsiella pneumoniae, Staphylococcus epidermidis and Escherichia coli were the most common isolated pathogens. S. maltophilia and E. coli were isolated from two UCB cultures compatible with the peripheral blood culture results, which shows that the bacteria were transmitted vertically from the mother to the neonate. In the present study, there was only one case of co-infection with E. coli and K. pneumoniae.A total of 22 bacterial isolates were traced in the neonates’ blood samples, in which S. epidermidis strains were susceptible to novobiocin in this study. A significant finding of the present study was the sensitivity of S. maltophilia strain to all the tested antimicrobial antibiotic agents.Fisher’s exact test showed that resistance to cefoxitin (P=0.01) was significantly greater in the Gram-positive than the Gram-negative bacteria; however, no significant differences were observed between the Gram-positive and Gram-negative bacteria in the patterns of resistance to cefixime (P=0.59), ceftriaxone (P=0.59), gentamicin (P=0.58), cefotaxime (P=0.59) and ciprofloxacin (P=0.59).2 test showed that the frequency of resistance to meropenem was significantly greater in the Gram-positive bacteria while resistance to amoxicillin clavulanic acid and ampicillin was significantly greater in the Gram-negative bacteria. Among the 22 isolates recovered from neonatal sepsis, 18 strains (81.8%) were Multi-Drug Resistant (MDR), containing 54.5% Gram-negative and 45.5% Gram-positive isolates. Fisher’s exact test did not show any significant relationships between the frequency of multi-drug resistance and bacterial Gram class (P=0.37).The χblaOXA48 and blaCTX-M-15 genes was found to be 25% and 50% in the Gram-negative isolate. As shown in blaOXA48 gene was traced in only three A. baumannii isolates, which also showed resistance to meropenem in the phenotypic analysis. The gene blaCTX-M-15 was detected in the Enterobacteriaceae species, which was consistent with their resistance to cefotaxime and ceftriaxone by the DDM method. In addition, the blaCTX-M-15 gene had the highest frequency among the K. pneumoniae isolates. These two genes were not detected among the S. maltophilia strains.The frequency distribution of blaCTX-M-15 gene is carried in the plasmid and is the most common β-lactamase leading to severe infection in infants (K. pneumoniae and E. coli isolates. The blaCTX-M-15 gene is an extended-spectrum beta-lactamase (ESBL) that is increasing in prevalence especially in Enterobacteriaceae  from LOS and one vancomycin resistant E. faecalis from EOS were isolated in this study. Rising VRE is a very alarming issue, since it has limited modes of therapy and causes irreparable damage, such as increasing the risk of mortality. In 2014, Shantala et al. reported a case of VRE in neonatal sepsis  vancomycin resistant l sepsis .K. pneumoniae, E. coli and coagulase negative staphylococci isolates in this research. Behmadi et al. reported coagulase-negative staphylococci were the most prevalent pathogens isolated from blood specimens in LOS and EOS  can offer a better understanding of the transmission of the pathogenic agents and analysis of the antibiotic resistance of them.blaOXA48 and blaCTX-M15 genes among bacterial isolates from a life-threatening infection such as neonatal sepsis is a huge concern and the selective pressure resulting from the excessive and empirical use of antibiotics in the community and hospitals leads to the replacement of the susceptible isolates with the resistant. The present study showed that Enterobacteriaceae and A. baumannii isolates were able to produce broad-spectrum β-lactamase and carbapenemases, respectively. Therefore, careful monitoring is necessary to detect and determine the antibiotic sensitivity of bacterial isolates in order to prevent the spread of these resistant genes among other strains through selecting the proper antimicrobial agent.The relatively high prevalence of


Mycobacterium avium complex (MAC) disease is difficult to treat with chemotherapy, and its mechanism of infection, infection route, disease onset, and severity remain unknown. Ficolins are oligomeric defense lectins. L-ficolin plays an important role in innate immunity. This study’s aim was to identify L-ficolin’s role in patients with pulmonary MAC disease.The incidence of infectious disease caused by nontuberculous mycobacteria is increasing worldwide. Pulmonary Between April 2011 and September 2017, 61 Japanese patients with pulmonary MAC disease were seen at our hospital. A control group, comprising 30 healthy individuals, without respiratory disease were enrolled in our study. The relationship between serum L-ficolin levels and disease severity was assessed, and L-ficolin’s antibacterial role was examined.p < 0.001). The cut-off value, based on receiver operating characteristic (ROC) analysis results, was 2.48 μg/ml (area under the curve (AUC) 0.90, sensitivity and specificity 83.6 and 86.7%, respectively). Serum L-ficolin levels were significantly lower in the patients with nodular bronchiectatic type disease compared with the patients with fibrocavitary type disease and were lower in the high-resolution computed tomography high-scoring group compared with low-scoring group. An in vitro analysis showed that purified recombinant L-ficolin bound to M. avium and its major cell wall component, lipoarabinomannan, in a concentration-dependent manner. In addition, recombinant L-ficolin suppressed M. avium growth in a concentration-dependent manner.Serum L-ficolin levels were significantly lower in patients with pulmonary MAC disease than in healthy subjects .This study is registered with UMIN ( Mycobacterium tuberculosis. However, chronic pulmonary disease, caused by NTM, is increasing in Japan because of an aging population. In 2014, the NTM incidence rate in Japan was 14.7 per 100,000 based on national surveillance data [Mycobacterium avium complex (MAC) organisms are the major causative bacteria of NTM infection in patients who do not have cystic fibrosis; these organisms are difficult to treat with chemotherapy. Pulmonary MAC disease is responsible for approximately 80% of the NTM cases in Japan. A systematic review indicated a high mortality rate among patients with pulmonary MAC [Nontuberculous mycobacteria (NTM) have been seen as environmental organisms of limited clinical relevance, overshadowed by the more aggressive Older age and female sex are well-known risk factors for NTM infection. Having a low body mass index (BMI) also appears to be a risk factor for NTM infection; higher BMI has a protective effect. Substantial heterogeneity was found among male patients, with the presence of cavity disease and high comorbidity levels predicting worse outcomes. In terms of molecular pathology, the adipokines leptin and adiponectin may be responsible for the presence of NTM disease –6. SeverInnate immune molecules can suppress the early stages of infection via mechanisms mediated by many different recognition systems and effectors, including the complement system. Pulmonary surfactant protein (SP-)A, SP-D, mannose-binding lectin (MBL), and ficolins are all types of complement lectins, capable of recognizing microbial carbohydrates and activating the lectin complement pathway via a mechanism similar to that of the classical pathway.Ficolins are oligomeric defense, proteins assembled from collagen-like stalks and fibrinogen-like domains that can sense danger signals, such as pathogens and apoptotic cells. Three members of the ficolin family have been identified and characterized in humans: M-ficolin (ficolin-1), L-ficolin (ficolin-2), and H-ficolin  –10. HumaHuman L-ficolin has lectin-like activity for N-acetylglucosamine (GlcNAc), lipopolysaccharides, 1,3-beta-D-glucan, lipoteichoic acid, and various acetylated compounds –13.L-ficolin’s fibrinogen-like domain forms a globular structure, similar to the carbohydrate-recognition domains of MBL, SP-A, and SP-D, and binds to sugar structures. Ficolins also show opsonic activity via the binding of their collagen-like domain to macrophages .Streptococcus pneumoniae, and M. tuberculosis), act as an opsonin, and promote microbe elimination through the elicitation of phagocytosis or the activation of the lectin pathway of the complement system in in vitro tests [L-ficolin is said to specifically bind to some clinically important microorganisms , and all participants gave written informed consent.Patients with NTM infection were included if they visited or were admitted to the hospital, and NTM infection was diagnosed based on the 2007 diagnostic criteria of the American Thoracic Society/Infectious Disease Society of America . ExclusiSerum L-ficolin levels were measured using an ELISA Kit  according to the manufacturer’s instructions.Within 3 months of entry into the study, thin slice 1 mm) HRCT images were obtained from the participants at 10 mm intervals from the lung apex to the base during suspended full inspiration with the participant in the supine position. All HRCT data were reconstructed using a high spatial frequency algorithm and displayed on the lung parenchymal window ; width, 1500 HU). The HRCT images were scored for the severity and the extent of the pulmonary MAC infection using the modified scoring system of Fowler et al. . The sum mm HRCT M. avium cultures were purchased from ATCC (No. 700898). This strain was the M. avium subspecies hominissuis, which is a major human MAC subtype in Japan [M. avium strain was clinically isolated from the sputum of an infected patient. The bacteria were cultured in MycoBroth and suspended in phosphate-buffered saline. The concentration of the bacterial suspension was determined by measuring the absorbance at 600 nm.MycoBroth and 7H11-C agar plates were purchased from Kyokuto Pharmaceutical . in Japan . AnotherM. avium was coated onto microtiter wells and then dried. After the wells were washed, the bacteria were incubated with L-ficolin at 37 °C for 1 h. The wells were washed and then incubated with anti-L-ficolin antibodies and secondary antibodies at 37 °C for 1 h. The binding of L-ficolin to M. avium was detected by measuring the absorbance at 492 nm.A suspension of UV-killed Sonicated lipoarabinomannan (LAM) was immobilized on an HPA sensor chip of the Biacore 3000 system  according to the manufacturer’s specifications. L-ficolin was injected at a flow rate of 30 μl/min. Sensorgrams of the interactions obtained using various concentrations of L-ficolin were analyzed using the BIAevaluation program.M. avium (2000 CFU) was cultured in MycoBroth at 37 °C for 96 h in the presence or absence of L-ficolin or other lectins. At 0, 8, 24, 48, and 96 h an aliquot of the culture was withdrawn and spread onto a 7H11-C agar plate. The plates were incubated at 37 °C for 48 h, and the number of colonies on the plates was then counted.All statistical analyses were performed using JMP 13.0  and GraphPad Prism v7 software . Student’s t-test or one-way analysis of variance (ANOVA) with post-hoc Tukey test were used to analyze continuous parametric data.p value < 0.05 was considered statistically significant.For all analyses, a This study included 61 patients with pulmonary MAC and 30 healthy volunteers in the control group. The participants’ characteristics are shown in Table 2, which was lower than that for individuals in the control group. Among the MAC patients, 43 (70.5%) had never smoked. Although white blood cell (WBC) counts were not significantly elevated, C-reactive protein (CRP) and serum SP-A, and SP-D levels were significantly higher in patients with MAC compared with those in the control group, suggesting an inflammatory reaction in the lungs of patients with pulmonary MAC disease.Patients with MAC were aged 70.9 ± 8.9 years on average and included 11 males (18.0%) and 50 females (82.0%); this was different from the control group but comparable to Japanese MAC surveillance data [M. avium , M. intracellulare , and mixed infections of MAC and other mycobacteria   Table . Anti-MAp < 0.001). A receiver operating characteristic (ROC) curve constructed between the MAC patients and healthy control participants revealed an area under the curve (AUC) of 0.90, a sensitivity of 83.6%, and a specificity of 86.7% when the cut-off for serum L-ficolin was set at 2.48 μg/ml . Compared with patients with the FC type, the patients with the NB type also showed lower levels of other parameters, such as WBC, CRP, albumin, and SP-A, indicating that the NB type could elicit a lower inflammatory response (Table p < 0.05).From the HRCT findings in MAC patients, the mean serum L-ficolin level in those patients with fibrocavitary type (FC type) was 3.09 ± 2.35 μg/ml, similar to the level seen in the healthy control group , of 8.96 × 10− 10 M. These results indicate that L-ficolin binds to M. avium and that the ligand targeted by L-ficolin might be LAM.We first confirmed the binding of recombinant L-ficolin to ner Fig. a. To detner Fig. b. The paM. avium in vitro , and indicated a negative correlation with the extent of the disease. Kitada et al. proved the usefulness of ELISA kits in diagnosing MAC . Higher neumonia . Patientneumonia , 30. InsAccording to the criteria for MAC, single and mixed mycobacterial infections were included, as shown in Table M. avium was specific but not completely dissociated by EDTA (data not shown). Previous reports indicated that the ligand for SP-D is LAM [M. avium when using ELISA methods (data not shown). As a result, we could not determine if there were one-to-one binding sites. Additional experiments to identify the ligand for L-ficolin will be necessary in the future.Ficolins bind to a variety of ligands on various pathogens. L-ficolin’s fibrinogen-like domain forms a globular structure, similar to the carbohydrate-recognition domains of MBL, SP-A, and SP-D, and binds to sugar structures. According to our binding analysis, L-ficolin’s binding to D is LAM . Our surM. avium growth without complement activation [M. avium growth. L-ficolin and SP-A form a bouquet-like structure consisting of six trimeric subunits. SP-D exhibits a cruciform structure consisting of four trimeric subunits. This structural difference causes distinct biological functions. Although L-ficolin was reported to bind to some microorganisms and act as an opsonin, direct inhibition of growth has not been reported. This novel effect of L-ficolin may play an important role in innate immunity in the pulmonary environment, especially via synergistic effects with SP-A.Previous studies have shown that complement lectins inhibit the growth of mycobacteria. Ariki et al. indicated that SP-A, but not SP-D, strongly inhibited tivation . We reveThere are some limitations to this study. First, the ages and sexes of the MAC group did not match those in the control group. Because the control subjects in this study were collected from health check individuals in a hospital, we could not match the status. Since previous reports indicated that the serum concentration of L-ficolin was not affected by age or sex, this mismatch is unlikely to have affected the study’s results –33. We cIn conclusion, insufficient serum L-ficolin is associated with disease presence and extent of pulmonary MAC disease. Serum levels of L-ficolin are a possible biomarker of pulmonary MAC disease.Additional file 1:Table S1A. is the summary of HRCT scoring system of the whole lung. To evaluate the disease progression of pulmonary MAC, we scored the HRCT findings. Each score was from 0 to 3 and the score of seven categories were totaled. The average HRCT score of each category was shown in table S1B. The average of total was 6.25 ± 3.30 indicating the patients’ severity were mild to moderate on the HRCT criteria.


The sensitivity, specificity, and positive and negative predictive values of TR-MRA compared to DSA were 100%, 97%, 73%, and 100%, respectively. Using the FWHM value to optimize the window level adjustment, the size of the residual component observed in the TR-MRA was larger compared to that in the DSA whereas the size of neck and the parent artery showed consistency between the two modalities. This image processing technique can be used as an effective screening tool for evaluating residual component in post-coiling brain aneurysms.Magnetic resonance image (MRI) is now widely used for imaging follow-up for post coiling brain aneurysms. However, the accuracy on the estimation of residual aneurysm, which is crucial for the retreatment planning, remains to be controversial. The purpose of this study is to evaluate a new post-processing technique that provides improved estimation of the residual aneurysm after coil embolization. One hundred aneurysms on 93 patients who underwent coil embolization for brain aneurysm were evaluated using the 1.5 Tesla time-resolved magnetic resonance angiography (TR-MRA) one year after the treatment. To minimize the inter-observer variability caused by the window level adjustment, an automatic post processing protocol using the full-width at half-maximum (FWHM) value was utilized. The result was then compared with that from the conventional cerebral angiography. Of the 97 aneurysms that underwent both TR-MRA and DSA, 23 (23.7%) showed residual neck / dome during follow-up. After window level adjustment, the size of the parent artery in the TR-MRA was consistent with that in the DSA. The reconstructed Volume Rendering images provided clear contours of the residual aneurysms and contributed to the understanding the configuration of residual aneurysm. The largest and the smallest diameter of the residual aneurysms was larger in the TR-MRA than in the DSA (8.05 vs. 7.72 mm,  Precise measurement on the “shape and size” of the residual component in a previously treated aneurysm is crucial in deciding for the re-treatment plan. Studies have indicated that time-resolved 3D contrast-enhanced magnetic resonance angiography (TR-MRA) is suitable for evaluating aneurysms previously treated with coil embolization or stenting , 2 becauThe full-width at half-maximum (FWHM) value, which is a commonly used measure in the field of electrical engineering, has been used for the quantitative measurements of images on computed tomography (CT) angiography or contrast-enhanced magnetic resonance imaging (MRI) to increase the accuracy of the quantitative data –5. The pFor qualitative analysis, detection sensitivity of the TR-MRA compared to the conventional DSA was evaluated. For quantitative analysis, measurement values of each residual aneurysm, as well as the size of parent artery, were compared between the two imaging modalities and statistical analysis was performed.Between July 2012 and March 2015, a total of 379 aneurysms were treated with coil embolization at the Jikei University Hospital. Of 287 patients with follow-up angiography after one year, 93 (100 aneurysms) who also underwent TR-MRA were included. The hospital’s institutional review board (IRB) approved the study.® . For those with wide neck aneurysms, the double catheter technique or balloon-assisted technique was primarily used, while the stent-assisted technique was selected as a rescue plan in case other techniques failed to prevent coil herniation into the parent artery. This work was approved by the ethics committee of the Jikei University School of Medicine. The approval number is clinical research 27–236 (8121). Both verbal and written consent was obtained for all patients before the treatment.Endovascular coil embolization of each aneurysm was done using the angiographic system, AXIOM Artis dBAOn the day of treatment, conventional 2D-DSA and 3D-DSA acquisitions were performed once the guiding catheter was positioned at the cervical segment of the internal carotid artery . For the acquisition protocol of the 3D-DSA, the motorized frontal C-arm, typically used for 3D rotational angiography, was used to acquire 496 projection images over a 200° arc  at 80 kV (peak). Moreover, 18 cc of diluted contrast material (60%) was injected at an injection rate of 3.0 cc/sec over 5 sec. The injection delay time was set to 1.5 seconds and the 3D-DSA was repeated at the end of treatment.®, Version 4.4.7.108.0 . Follow-up DSA was repeated 12 months after the treatment as a routine procedure, using the same acquisition protocol.The acquired images were further processed by an image-processing application, Aquarius iNtuition®; Siemens Healthcare GmbH, Erlangen, Germany), with a 4-channel head coil. This was repeated at 3 and 6 months post-treatment. Subsequently, TR-MRA was performed at approximately one year after the treatment. The imaging parameters used for the acquisition were as follows: TR/TE, 2.36 msec / 0.94 msec; flip angle, 30°; FOV, 220 mm; matrix, 224 x 175; slice thickness, 1.0 mm; bandwidth, 970 KHz; and the parallel imaging method with k-space sensitivity encoding “GRAPPA” , 2. A coronal or a sagittal 20-mm slab was acquired, depending on the relation between the parent artery and the aneurysm.Routine post-treatment MRI, including TOF MRA, was performed one day after using the 1.5T MR imaging system  of contrast medium: 0.2 mL x body weight of Gadodiamide Hydrate . This was injected at a flow rate of 2 mL/sec, followed by a saline flush of 20 mL. The TR-MRA was initiated 8 sec after the start of injection of the contrast medium.Ten dynamic scans were obtained at a temporal resolution of 1.23 sec, with voxel size of 1.0 x 1.0 x 1.0 mm and total scan time of 40 sec. A monophasic injection protocol was implemented, with automatic power injection  performed the qualitative analysis to evaluate whether the image findings in the reconstructed TR-MRA images, which was performed 1 year after the treatment, were consistent with the 1 year follow-up DSA findings. The evaluators were independent from the decision making of the treatment planning of each patient, thus the evaluations were performed in the blind fashion.A dataset of anonymized and randomly numbered TR-MRA images was created for the evaluation. Another dataset for DSA images was also created in the same manner. Two neuro-intervetionalits reviewed each dataset individually and assessed using the following classification. The “Major re-canalization” was defined as “Raymond-Roy Occlusion Classification 3 and the observer recommended re-treatment based on the image findings” . Each anThe sensitivity, specificity, positive predictive value, and negative predictive value, as well as the 95% confidence interval (95%CI) were calculated. The inter-modality agreement was evaluated using kappa statistics to investigate the inter-rater reliability between the two evaluators.Patients who were found to have a residual neck/aneurysm on one-year follow-up DSA or MRA were selected for the analysis. The size of the residual aneurysm was calculated as follows. First, a 2D DSA image with optimal projection for intra-operative view was selected and a line connecting the most proximal and most distal points of the aneurysm neck was drawn (neck line). The largest and smallest diameters of the residual aneurysm in the same projection was then measured and recorded. The same parameters of the reconstructed TR-MRA image using the same projection were evaluated in the same manner. Lastly, to confirm inter-modality consistency, the size of the parent artery at the proximal neck was compared between the TR-MRA and the 2D DSA.The measurement values of each residual aneurysm as well as the parent artery were compared between the TR-MRA and 3D-DSA images. The between-group correlation was analyzed using Spearman’s rank correlation coefficient (Spearman’s Rho). To examine differences in size measurements between the two groups, statistical analysis using Wilcoxon signed-rank test was also performed.One hundred aneurysms in 93 patients  successfully underwent coil embolization. The location of the aneurysms were the anterior communicating artery (n = 15), anterior cerebral artery (n = 2), middle cerebral artery (n = 15), cavernous internal carotid artery (ICA) (n = 3), para-clinoid ICA (n = 10), ophthalmic artery (n = 11), ICA superior hypophyseal artery (n = 16), posterior communicating artery (n = 12), anterior choroidal artery (n = 4), intracranial vertebral artery (n = 1), basilar trunk (n = 4), and basilar top (n = 7). Both the TR-MRA and DSA were performed approximately one year after the treatment in all of the patients.Of the 100 aneurysms, 97 were included in the study after three were excluded due to sub-optimal contrast filling in the TR-MRA. The average maximal diameter of the aneurysms was 6.9±3.2 mm before the treatment. Twenty-one aneurysms (21%) were treated by stent-assisted coil embolization, while the rest were treated by the simple technique, double-catheter technique, or balloon-assisted technique. The mean follow-up period after coil embolization was 16.3 and 15.9 months for the DSA and TR-MRA, respectively.The VR images of TR-MRA were successfully created in all 97 aneurysms. The typical VR images of TR-MRA and DSA of post-coiling aneurysms were shown in Figs Overall, the reconstructed VR images provided clear contours of the residual aneurysms and contributed to the understanding of the projection, shape, and size of the residual component.The results showed that the sensitivity, specificity, positive predictive value, and negative predictive value of TR-MRA were 100% (95%CI: 63–100%), 97% (95%CI: 90–99%), 73% (95%CI: 39–94%), and 100% (95%CI: 96–100%), respectively . The unwThe 23 aneurysms (23.7%) diagnosed with residual neck/aneurysm on follow-up DSA or MRI were selected the quantitative analysis. First, to evaluate the correlation between the two groups, spearman’s rank correlation coefficient was used for statistical analysis. The largest diameters and smallest diameter in the TR-MRA and the DSA were strongly correlated (Spearman’s rho = 0.962 and 0.746 respectively). Similarly, the neck diameters and parent artery were strongly correlated (Spearman’s rho = 0.980 and 0.896 respectively). .p = 0.0004 and p = 0.007 respectively). On the other hand, mean value of the parent artery in TR-MRA was 2.75 mm  for TR-MRA and 2.66mm (95% CI: 2.31–3.01mm) for DSA. Similarly, the neck size in TR-MRA was 6.82 mm  for TR-MRA and 6.29 mm (95% CI: 4.98–7.6mm) for DSA. Both parameters showed statistically no significance (p = 0.260 and p = 0.069 respectively)Second, to examine differences in size measurements between the two groups, the statistical analysis was performed using the Wilcoxon signed-rank test. The summary of the quantitative analysis was shown in The method of imaging follow-up for aneurysms treated with endovascular technique remains debatable –8]. Alt. Alt8]. Studies have indicated that TR-MRA reduced influence from aforementioned imaging artifacts compared to TOF-MRA . HoweverQuantitative analysis revealed that the size of the residual component determined by TR-MRA was slightly larger than that by DSA despite the size of parent artery showed consistency between the two modalities. It is potentially because TR-MRA is known to have less susceptibility artifact induced by the metal implant as compared to other MRI sequences , whereasNonetheless, these findings are corroborated by the qualitative analysis, which demonstrates that TR-MRA can detect “major recanalization” with 100% sensitivity and 97% specificity. There were some false positive (3%), but no false negatives (0%), indicating that this imaging method may overestimate, but quite unlikely underestimate, the volume of residual aneurysm compared to DSA. Therefore, this imaging modality can be an effective screening tool for detecting re-canalization of a treated aneurysm before considering DSA.®, is characterized by its unique rendering method called the “Image-Order Rendering”, which is designed to prevent the degradation of the image quality during the 3D reconstruction process and provide clearer images. The reconstructed VR images showed the clear contours of the artery, thus the shape and projection of the residual aneurysm were clearly visualized. This helps evaluators assess the configuration of residual component in each treated aneurysm.It should be noted that the 3D workstation used in this study, Aquarius iNtuitionThe TR-MRA, however, has its own limitations. For instance, the signal intensity of small / peripheral vessels tends to decrease compared to those of other sequences like TOF. Insufficient signal-to-noise ratio can lead to image degradation. Moreover, inappropriate selection of the time-phase to reconstruct the 3D images can also lead to image degradation. Lastly, the current study was performed using a 1.5 T MRI. Using an MRI with a stronger magnetic field, e.g. 3.0 T, can increase the signal-to-noise ratio and may solve part of the limitations . FurtherA linear contrast agent was used when this study was conducted. However, with the increasing reports about their side effects and the accumulation in the body , we beliUsing the full-width at half-maximum (FWHM) value to optimize the window level adjustment, the reconstructed TR-MRA images provided improved visualization with consistent measurement values of the residual aneurysm. The size of the residual component observed in the TR-MRA was larger compared to that in the DSA whereas the size of neck and the parent artery showed consistency between the two modalities. This image processing technique can be used as an effective screening tool for evaluating residual component in post-coiling aneurysms.S1 TableA. Residual aneurysm measured by TR-MRA: The measurement values of three parameters 1) Neck length, 2) Largest diameter, 3) Smallest diameter as well as the volume of residual aneurysm, which was calculated by the image-processing application “Aquarius iNtuition®” are listed. B. Residual aneurysm measured by DSA: The measurement values of the same parameters described above were listed. C. Diameter of the parent artery: The size of the parent artery at the proximal neck was compared between the TR-MRA and the 2D DSA.(PDF)Click here for additional data file.


Arthrospira platensis is the widely available source of spirulina that contains distinctive natural pigments, including carotenoids and C-phycocyanin (C-PC). In this study, the major carotenoid and C-PC contents were determined in seven commercially available spirulina powder products and laboratory-prepared A. platensis trichomes (AP-1) by an LC-DAD method and UV-Visible spectrometry, respectively. The correlation of these two pigment content levels with Hunter color coordinates and antioxidant activity was also evaluated. The L* value failed to show a significant correlation with pigment content, but a positive correlation was observed between a* values and the contents of total carotenoid and C-PC. As b* values decreased, the chlorophyll a and C-PC contents increased. AP-1 exhibited the highest content of total carotenoids, chlorophyll a and C-PC, and antioxidant activities among the samples. This observation could be related to degradation of these pigments during the mass production process. The carotenoid profiles suggested that the commercial spirulina powders originated from two different sources, A. platensis and A. maxima. Total carotenoid and C-PC content exhibited positive significant correlations with antioxidant activities measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. These results provide a strong scientific foundation for the establishment of standards for the commercial distribution of quality spirulina products. Arthrospira (Spirulina) platensisis or A. maxima [Most commercial spirulina powder is composed of dried bodies of . maxima . These s. maxima ,3, spiru. maxima .Carotenoids are a class of natural lipid-soluble pigments that are responsible for the red, yellow, and orange colors found in various plants and microorganisms. They function primarily as photosynthesis aids and are used in the photoprotection process. Humans and other animals are unable to synthesize carotenoids and acquire them through alimentation. Carotenoids are used in food and feed as colorants and flavorings, and in nutritional supplements as a source of provitamin A . The heaC-Phycocyanin (C-PC) is a hydrophilic and intense blue-colored biliprotein found in blue green algae. C-PC comprises a protein and the chromophore phycocyanobilin. The protein moiety consists of α and β subunits with molecular weights near 18,000 and 20,000 Da, respectively. This colorant is highly stable in the pH range of 5–8 and exhibits a strong red fluorescence when present in its native form . The bioA. platensis trichomes cultured in our laboratory which could give best condition for retaining these pigments. Additionally, the correlation of pigment levels with the Hunter Lab color parameters was assessed as was the relationship of the pigments to antioxidant activity as a method to better understand predictive quality factors for these ingredients.These pigments can be easily degraded during the mass production process using sunlight or hot-air dry. In order to provide improved characterization of available spirulina ingredients to manufacturers and scientistz, the carotenoid and C-PC content levels were determined for seven commercially available spirulina powder products and for freeze-dried Athrospira platensis cultivated in our laboratory (AP-1) or purchased from on-line and local stores (C1–C7). All of the powders were dark green and differed slightly from each other in the Hunter color coordinates of brightness (L*), redness (a*) and yellowness (b*).Because carotenoids and C-PC are pigments, a colorimetric evaluation was performed on the spirulina powders obtained from L* and b* values were obtained in C6, with C7 displaying the second highest L* value . The limits of detection (LOD) were determined by serial dilution based on a signal-to-noise (S/N) ratio of 3:1 . AP-1 showed the greatest amount of C-PC, 251.2 ± 11.2 mg/g, and the mean values in the commercial spirulina samples were between 94.9–153.3 mg/g .w/w). The average percentages of chlorophyll a and carotenoids were 0.26–1.1% and 0.03–0.38%, respectively. The total carotenoid content varied by up to eight-fold among the commercial samples, while the content variations of chlorophyll a and C-PC were 1.8 and 1.6-fold, respectively.Among the three major pigments in spirulina, the content of C-PC was highest with a value of 10–25%  and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay methods were applied to determine the antioxidant activity of the carotenoid and C-PC extracts obtained from our spirulina samples. The C-PC extract showed higher antioxidant activities than the carotenoid extract in the DPPH and ABTS assays . The carThe total phenol and flavonoid contents were evaluated to investigate the effect of phenolic and flavonoid compounds on the antioxidant activities of these extracts. The results showed that the mean value of total phenol content in the carotenoid extract was in the range of 1.3–6.4 μmol gallic acid equivalents (GAE)/g dry weight, which was much lower than that of 47.0–82.1 μmol GAE/g observed in the C-PC extract . AdditioA. platensis trichomes cultured in our laboratory (AP-1). The correlation of the content level of these two pigments with a visible parameter and antioxidant activity was also evaluated.Spirulina has been used as an additive in a variety of health foods and animal feeds, and has been produced commercially for the last 30 years for these purposes. Commercially grown spirulina is normally produced in large outdoor ponds under controlled conditions or harvested directly from lakes . It contL* value failed to show a significant correlation with pigment content or antioxidant activities  increased, the total carotenoid and C-PC content increased. The redness value displayed a non-significant correlation with chlorophyll a content. As the b* values (+yellow −blue) decreased, the chlorophyll a and C-PC content of the extracts increased. Although dark green color of chlorophylls is dominant in the spirulina powders, this result suggests that the redness and blueness would reflect the content of carotenoids and C-PC.The tivities . As the trans-β-carotene was highest among the four major carotenoid ingredients of the spirulina powder samples in AP-1, C1, C2 and C5 while that of all-trans-zeaxanthin was highest in the other samples. The other four samples showed lower a* values than the formers. Spirulina are multicellular and filamentous blue-green microalgae belonging to the two separate genera—Spirulina and Arthrospira—which consist of approximately 15 species. Of these, Arthrospira platensis is the most common and widely available source of spirulina and most of the published research and public health decisions refer to this specific species [A. plantensis was chosen and cultivated in this study as a control. However, two Arthrospira species, A. platensis and A. maxima, have been used as a food source, dietary supplement, and feed supplement [A. maxima sample [A. platensis and the other group belonged to A. maxima. Contrary to previous findings, β-cryptoxanthin was not detected in the spirulina powder used in this study [cis-β-carotene and 13-cis-β-carotene . AP-1 was cultivated and freshly prepared in our laboratory. The total carotenoid, chlorophyll a and C-PC contents observed in AP-1 were the highest seen among the eight samples. AP-1 also exhibited the highest antioxidant activities among the samples. The overall lower amounts of carotenoid, chlorophyll a and C-PC contents found in commercial spirulina powder compared to AP-1 could stem from pigment degradation during the mass production process, as the natural pigments are sensitive to light, heat and oxygen. It has been reported that the carotenoid composition differences of dried commercial spirulina preparations are most likely due to their thermolability . While robserved . Chloropobserved . A solarnd light .Total carotenoid content showed high correlation coefficients of 0.92 and 0.86 to chlorophyll a and C-PC content, respectively. The total carotenoid content also exhibited greater positive correlations with antioxidant activities than the total phenol or flavonoid contents of the carotenoid extract did. These strong correlations suggest that the major antioxidant compounds in the carotenoid extract are carotenoids, not phenols or flavonoids, although phenols and flavonoids are also known antioxidants, and the flavonoid content level contributed in portion to the antioxidant activity of the samples with low content of total carotenoids. Chlorophyll a and C-PC contents also showed significant positive correlations, respectively, with the other two pigment contents and antioxidant activities. Different from the carotenoid extract, the total phenol content in C-PC extract showed similar positive correlations with antioxidant activities of this extract compared to the C-PC content in C-PC extract. This result suggests that the phenol compounds in the C-PC extract also contributed to the antioxidant activity in portion, although C-PC is the highest abundant and major antioxidant ingredient of this extract.The antioxidant activities against DPPH and ABTS radicals displayed overall significant positive correlations to each other in both carotenoid and C-PC extracts. While in the carotenoid extract DPPH assay exhibited greater correlations with the lipophilic total carotenoid, chlorophyll a and flavonoid contents than ABTS assay, in the C-PC extract ABTS assay showed higher positive correlations with the hydrophilic C-PC and total phenolic contents than DPPH assay. This result is well consistent with the previous report that the high-pigmented and hydrophilic antioxidants are better reflected by ABTS assay than DPPH assay .This study revealed that the color attribute of spirulina powder, redness and blueness, would be a factor to assume the lipophilic carotenoid and hydrophilic C-PC contents, and the powder with high level of carotenoid content has also high possibility to show high levels of chlorophyll a and C-PC contents with high antioxidant activity. In addition, the carotenoid profile can be utilized as a parameter for the discrimination of botanical source. These results provide a scientific foundation for proper identification and establishment of standards for distribution of quality commercial spirulina products. Future studies will evaluate the stability of these natural pigments in spirulina powder during storage under various conditions.Athrospira platensis (Nordstedt) Gomont (P_PS_00001194) was provided by the Korea Institute of Ocean Science and Technology . Cultivation was conducted for a period of 10 days in 500 mL baffled flasks containing 300 mL of Zarrouk’s medium  with stirring at 120 rpm at 25 °C under 130 μmol/m2 s illumination. Cells were harvested by centrifugation  at 3000× g for 5 min at 4 °C and freeze-dried . The lyophilized sample (AP) was stored at –80 °C until analysis was completed. The seven commercially available spirulina powder samples (C1–C7) were purchased from various online and in-store sources from November to December in 2017. The cultivation regions of these products included Australia , China , India , New Zealand  and USA .L* (lightness), a* (redness), and b* (yellowness) values according to the Hunter color system.Color attributes of samples were measured with a colorimeter . Prior to testing, the colorimeter was calibrated using a Minolta standard white reflector plate. Color measurements were taken between three and five times per sample, depending on the portion of each powder. The data were presented as 2SO4 and NaHCO3. The solution was transferred to a 10 mL conical tube and sonicated three times for 10 min. The extract was centrifuged at 5700× g at 4 °C for 10 min , and 5 mL of the supernatant was dried under a stream of N2 gas and dissolved in 500 μL of a CH2Cl2 and acetone mixture . This sample solution was filtered through a 0.45 μm membrane filter (polytetrafluoroethylene (PTFE), 13 mm, 0.45 μm, Whatman, Maidstone, UK) prior to LC analysis.All extraction procedures were performed under subdued light to avoid pigment degradation. Two hundred and fifty milligrams of the lyophilized samples were homogenized using a pestle in a prechilled mortar with 1 mL of acetone , sea sand, NatR (min): 25.0 for all-trans-zeaxanthin, 27.2 for chlorophyll a, 28.9 for diatoxanthin, 36.3 for 13-cis-β-carotene, 39.0 for all-trans-β-carotene and 40.3 for 9-cis-β-carotene. Methanol, water and methyl-tert-butyl ether used in the HPLC system were all of HPLC grade, and all other chemicals were extra grade. A stock solution of diatoxanthin (0.691 mg/L) was purchased from DHI , and the other carotenoids and chlorophyll a were purchased from CaroteNature GmBH  and Sigma, respectively.Carotenoid analysis was conducted according to our previously reported method  using ang at 4 °C for 6 h and the supernatant was filtered through a 0.45 μm membrane filter . Two hundred microliters of each extract was transferred to a 96 well plate, where a microplate reader  was used to detect the absorbance at 620 nm. Working calibration solutions in the range of 0.5–5 mg/mL were prepared by diluting the stock solution of C-phycocyanin  with PBS buffer. All of the procedures were performed under subdued light to avoid pigment degradation as described above.C-PC was quantified by UV-Visible electronic absorption using an external calibration method . BrieflyThe total phenol content of the extract was determined spectrophotometrically according to our previously described method , which iThe total flavonoid content was determined using a diethylene glycol colorimetric method  using quThe scavenging activities of the samples on 1,1-diphenyl-2-picrylhydrazyl (DPPH) were measured using a slightly modified form of our previously reported method . The rad•+ solution at room temperature, and the absorbance was measured at 734 nm after 10 min. The antioxidant activity of each sample was expressed as Trolox (Sigma) equivalents per gram (μmol TE/g).The ABTS radical was generated using a previously reported method . Each exAll of the contents and the antioxidant activities are expressed as the means ± standard deviations (SD) of triplicate determinations. The differences among samples were statistically evaluated via one-way analysis of variance (ANOVA). The values were evaluated at the 5% significance level using two-sided tests. Pearson’s correlation coefficients were obtained using IBM SPSS Statistics 24.0 software .


Olive polyphenols exert many favorable properties, such as an antioxidative effect in humans, but are poorly studied in animals, especially in pigs. Therefore, we conducted a trial including olive leaf extract (OLE), which is rich in polyphenols, in pigs fed a linseed oil-enriched diet, which is a known cause of postprandial oxidative stress. OLE exerted some antioxidative effects in piglets fed a polyunsaturated fatty acid (PUFA)-rich diet without any negative effects, even when included in a higher concentration than that proposed for humans. Additionally, the inclusion of OLE did not compromise the zootechnical performance of the piglets as in the case of the dietary inclusion of olive leaves. Therefore, OLE could be used as an effective feed additive for beneficial effects on intestinal health and meat quality, among others.2-isoprostanes, as well as of plasma malondialdehyde and γ-tocopherol, but raised the plasmatic α-tocopherol and altered the level of urinary 8-OHdG. In conclusion, only minor positive effects of dietary OLE on the oxidative stress parameters were observed. Additionally, OLE did not show concentration dependence. The aim of the current study was to assess the antioxidative effects of the dietary supplementation of olive leaf extract (OLE) in different concentrations compared to those of vitamin E in piglets under conditions of dietary n-3 PUFA-induced oxidative stress. Forty-eight castrated male piglets (10.6 ± 0.99 kg) were fed the following experimental diets: Cont− , Cont+ , Vit-E , OLE-1 /day), OLE-2 , and OLE-3 . After 21 days of feeding, the experimental diets, blood and urine samples were collected to assess the extent of the oxidative stress. Results indicated that diet OLE-1 lowered the activity of gamma-glutamyl transferase, protected DNA (measured as DNA tail %) and altered urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG). Dietary vitamin E lowered the levels of urinary F Olea europaea L.) contains different polyphenols, of which some are suggested to have antioxidative and other beneficial properties in humans [Olive plant  as a source of polyphenols, and to compare them to the effects of a supranutritional vitamin E supplementation in piglets. In order to measure the possible antioxidant effects of olive polyphenols, the oxidative load was increased via a PUFA-rich diet, known to induce postprandial oxidative stress in pigs, and therefore an additional supplementation with antioxidants is beneficial . Since oForty-eight weaned and castrated commercial male piglets  were used in the trial, which was divided into two repetitions with twenty-four animals. The piglets (10.6 ± 0.99 kg) were assigned to six experimental groups and fed the experimental diets once per day for 21 days. The animals were caged individually in balance cages (850 mm × 530 mm × 760 mm), which permitted the individual collection of urine samples. The temperature was kept at 28 °C in the beginning and was gradually lowered to 24 °C, while light was provided for 13 hours per day (07:00–20:00 h). The experimental design and protocol were approved by the Animal Ethics Committee of the Veterinary Administration of Slovenia (U34401-9/2016/7).The experimental feed was formulated according to the Nutrition requirement of swine  and was Olive leaves collected from an organic olive grove were dried at 45 °C, milled , immersed in 70% ethanol (250 g leaves/L) and shaken on an orbital stirrer for four days. After extraction, the mass was filtered through a gauze and evaporated at 40 °C to 15% of the initial weight. Then, 20% propylene glycol:water (1:1) was added in order to dissolve the residuals. The final extract contained 1.70% oleuropein, 0.64% hydroxytyrosol and 5.85% total polyphenols , determined according to the official method of the International Olive Oil Council (COI/T.20/Doc no. 29). Hydroxytyrosol equivalents (EQs) were calculated as the sum of (free) hydroxytyrosol and hydroxytyrosol contained in oleuropein, using the stoichiometric ratio between the two.At the end of the trial, blood from the jugular vein was collected into Vacuette tubes . The plasma was collected in K3-EDTA tubes and centrifuged (1800 g for 15 min at 4 °C) to conduct the analysis of MDA, α- and γ-tocopherols. Whole blood was used to conduct the analyses of hemoglobin and comet assay. The serum for the analyses of the antioxidant capacities of water (ACW)- and lipid (ACL)-soluble antioxidants, activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT), as well as oxidized LDL (oxLDL), was taken into collection tubes with a clot activator, left for 2 h at room temperature and centrifuged at 2500 g for 10 min at 20 °C. Lithium heparin tubes were used to collect whole blood for the analyses of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). 2-isoprostanes and 8-OHdG, respectively, was performed prior to storage at −80 °C.Urine samples were collected from days 17 to 19 (48 h) of the experimental period. Urine samples were collected in polypropylene containers on a daily basis and stored at 4 °C. Total collected urine samples (48 h) were weighted, filtered through a filter paper, purged with a nitrogen stream and stored in polypropylene micro-centrifuge tubes. The addition of 0.005% of butylated hydroxytoluene and 0.05% sodium azide for the analyses of F2-isoprostanes (iPF2α-III) were measured by a commercial ELISA kit , and 8-OHdG by the ELISA kit ADI-EKS-350 . The fatty acid profile was measured as described by Frankič and Salobir [Feed samples were analyzed weekly for dry matter, crude protein, crude fat, crude ash, acid and neutral detergent fiber (ADF and NDF), Ca and P using the methods of Naumann and Bassler . Plasma  Salobir .p < 0.05 , and the actual P-value is given for tendencies. For the analyses of variance, the mixed procedure from SAS software was used . In the statistical model, the experimental groups, the replicates and the interaction between the two were used as fixed effects, while an individual animal was considered as an experimental unit. The repeated statement was used to include the analytical variability within an experimental unit. For the analyses of the results of the comet assay, the random statement was used to include the measured values of the cells within an experimental unit. The heterogeneous variance was presumed in the observed parameters and differences between the groups were tested with the Tukey–Kramer multiple-comparison test. The least square means and the SEMs as a dispersion parameter are shown in the results section. Statistical significance was accepted at p > 0.05) in the final body weight or body weight gain were obtained. Animals fed Cont− had higher (p < 0.05) feed intake and poorer conversion ratio in comparison to the groups fed linseed oil-supplemented feeds  was detected, while no differences were obtained between the other groups. Regarding plasma MDA levels, they were higher in piglets fed Cont+ (p < 0.05) than in piglets fed Cont−. The latter, in turn, had lower (p < 0.05) plasma MDA levels than OLE-1, OLE-2, and OLE-3. A statistical tendency (p = 0.104) was obtained between the plasma MDA levels of animals fed Vit-E and those of animals fed Cont+, with the first being lower. Piglets fed Vit-E also had lower plasma MDA levels than those fed OLE-1 (p < 0.05). The supplementation with OLE did not affect plasma MDA levels in comparison to Cont+. In relation to the serum oxLDL concentration and urinary MDA excretion levels, no differences were observed between groups, although statistical tendencies were obtained between the urinary MDA excretion levels of piglets fed Cont− and those fed Cont+ (p = 0.115) and OLE-3 (p = 0.074) (Lower urinary excretion of F= 0.074) , the firp < 0.05) in comparison to these groups. Piglets fed Cont− had a lower level of plasma γ-tocopherol than those fed Cont+, while piglets fed Vit-E had a lower level of plasma γ-tocopherol than those fed Cont−, Cont+ and OLE-supplemented diets. By means of the determination of the ACW, we observed a tendency of animals fed Cont− to have a lower ACW than those fed OLE-2 (p = 0.078). Analyses of the GPx activity resulted in higher GPx activity in animals fed Vit-E than those fed OLE-1 (p = 0.096) and OLE-2 (p = 0.083). Serum ACL and SOD activity did not differ between the groups  tail DNA % than those fed Cont+ and Vit-E, but not than those fed the OLE-supplemented feed, regardless of the inclusion rate. Regarding the OTM, the only difference obtained was the higher value observed in piglets fed Vit-E (p < 0.05) compared to those fed Cont−, Cont+ and OLE-supplemented feeds. Urinary 8-OHdG excretion was higher (p < 0.05) in animals fed Cont− than in those fed Cont+, OLE-2 and OLE-3. Moreover, piglets fed Vit-E had a higher excretion of 8-OHdG (p < 0.05) than those fed Cont+, OLE-2 and OLE-3. However, the 8-OHdG excretion of pigs fed the OLE-supplemented diet did not differ from that of piglets fed Cont+. Serum GGT activity was lower in animals fed Cont− and OLE-1 than in those fed Cont+ (p < 0.05), while there were no effects on serum GGT activity of animals fed Vit-E, OLE-2 and OLE-3. No differences in serum AST and ALT were found between the groups  in comparison to Cont− (5.3:1). The differences in the dietary linoleic fatty acid, which is elongated to arachidonic fatty acid, the precursor of F2-isoprostanes, were reflected in the fatty acid composition of the erythrocytes. Animals in group Cont− had a higher proportion of arachidonic acid in the erythrocytes  than the linseed oil-supplemented groups , and therefore higher F2-isoprostane levels in Cont− were expected. Therefore, it is not clear if the differences were due to the difference in the level of arachidonic acid in the body or to the lower oxidative stress in animals fed Cont− than in those fed Cont+.One of the most accepted markers of in vivo oxidative stress is urinary excretion of Frostanes . We did n humans . In the n humans . F2-isopPrevention of the oxidation of LDL is an important effect of olive polyphenols in protection against cardiovascular diseases in humans. In the present study, the plasmatic level of oxLDL was not affected by any of the supplements used compared to both Cont− and Cont+. This does not comply with the results reported by Paiva-Martins et al. , who obsp < 0.05) and showed a tendency to increase urinary MDA excretion levels (p = 0.115). This is in accordance with the previous findings of Frankič [Dietary linseed oil (Cont+ vs Cont−) increased plasma MDA levels , although the effect was not present in higher concentrations of OLE (OLE-2 and OLE-3). This partly agrees with some in vitro [The oxidative products of n-3 PUFA can form adducts with DNA, suggesting their involvement in the pathogenesis of different diseases . In our in vitro ) and in in vitro  studies in vitro . These aThe urinary level of 8-OHdG is a common marker of DNA damage or its repair . In the p < 0.05 and p = 0.078, respectively) than those fed Cont−. This could indicate protective effects of OLE-1 and Vit-E, as demonstrated for other dietary antioxidants [p > 0.05). In the present study, the activities of AST and ALT were not influenced by any of the supplements used, which is in agreement with the study by Bock et al. [Lipids are primarily metabolized by the liver, and therefore its enzymes are an important marker of oxidative stress caused by n-3 PUFA. In our study, the inclusion of linseed oil (Cont+ vs. Cont−) increased the GGT activity in serum, suggesting an effect of the high intake of PUFA on liver function. Animals fed OLE-1 and Vit-E had lower GGT activity (oxidants . Howeverk et al. . These aAntioxidants commonly have a concentration-dependent effect on the prevention of oxidative damage. Thus, we hypothesized that this could also be the case for OLE. Even though there are some reports on the concentration-dependent effects of olive polyphenols in pigs , turkeysIn conclusion, the lowest level of the dietary OLE affected n-3 PUFA-induced oxidative stress measured as 8-OHdG excretion, DNA tail % and liver GGT in piglets. The supranutritional level of vitamin E did not show clear effects on lipid peroxidation and DNA damage, even though positive effects on the excretion of 8-OHdG and on the hepatic GGT were observed. Despite some positive effects of OLE on oxidative stress reduction, the results did not show clear concentration dependence and should be further tested to obtain optimal dietary concentrations. Since growth and metabolic rate are age dependent, the effects of OLE might be stronger in older animals, a longer supplementation period, and/or ad libitum feeding.